This grant application is a competitive renewal of an RO1 from a highly productive and well-established investigator that will extend his previous studies on the development and application of novel isoform-selective nitric oxide synthase (NOS) inhbitors.Nitric oxide (NO) is produced during the oxidation of L-arginine to L-citrulline by NO synthase (NOS), and plays a critical role in mediating cellular function in diverse physiologic settings. Three NOS isoforms have been identified, including endothelial (eNOS; or type III), neuronal (nNOS; or, type I) and inducible (iNOS; or, type II) NOS. Although NOS has been shown to contribute to normal physiologic functions, including the regulation of vascular tone, neurotransmission, and others, excessive NO production can also have pathophysiologic effects. Of the 3 NOS isoforms, iNOS has been most consistently identified as contributing to high rates of NO production in pathologic settings, including septic shock, vascular edema, tissue injury due to inflammation, tumor angiogenesis, and other diseases. In several animal models of these disorders, treatment with isoform-selective NOS inhibitors have been shown to modulate disease severity, suggesting a potential therapeutic role of these agents in the clinical arena. One of the major limitations in the clinical use of NOS antagonists, however, is that non-selective inhibition of NOS may disrupt normal physiologic function and have adverse effects. As a result, there is a critical need for the development of highly specific, isoform-selective NOS inhibitors. The PI proposes to design, synthesize and test new NOS inhibitors that selectively target the iNOS and nNOS isoforms. The basic hypothesis is that "better NOS inhibitors can be more efficiently designed using recently developed information on the structure of NOS isoforms, on the NOS reaction mechanism, and on the interactions of NOS with its cofactors, substrates and products." These studies will primarily focus on analogs of L-arginine since it is readily transported into cells and can incorporate structural modifications that achieve isoform selectivity in binding and susceptibility to catalytic activation. The design of these inhibitors will incorporate new information on mechanisms of NOS activity and substrate specificity of the different NOS isoforms, as well as recently published data on NOS structure from x-ray crystallography. The PI proposes to test these new inhibitors by studying their effects on human NOS isoform binding and activity in vitro for selectivity, and if successful, these agents will be further studied with rodent models in vivo. This proposal includes 3 specific aims. In aim #1, the PI will determine mechanisms by which vinyl-L-NIO, a NOS inhibitor that was previously designed and tested by the PI, selectively and irreversibly inhibits the nNOS isoform. Based on these studies of mechanisms underlying relative nNOS selectivity of vinyl-L-NIO, the PI will design analogs of vinyl-L-NIO that may have even greater nNOS selectivity. In Aim #2, the PI will screen known and new compounds that can inhibit both NO and superoxide generation by NOS. In Aim #3, new isoform-selective L-arginine antagonists will be developed and studied in an attempt to develop agents with preferential binding to the iNOS isoform. Although initiated during the previous grant period, the development of these agents met with limited success. The current strategy is to extend these studies by utilizing new information from high resolution x-ray crystallography studies on eNOS and iNOS isoform structure. The PI will also exploit new agents from Glaxo-Wellcome, sulfoximine-based iNOS -selective inhibitors, to further develop new analogs of these agents.